The present invention relates to nucleic acid sequences having promoter activity, which cause a leaf-specific expression, in plants, of encoding nucleotide sequences under the control of said promoters, to expression cassettes, recombinant vectors and microorganisms which embrace such regulatory sequences, to transgenic plants transformed with them, to a method of producing transgenic plants, and to a method of isolating the leaf-specific promoter.
It is known to transfer foreign genes into the genome of a plant in a targeted manner by means of genetic engineering methods. This process is termed transformation, and the resulting plants are termed transgenic. Transgenic plants are currently employed in various fields of biotechnology. The predominant aims are, on the one hand, crop protection and, on the other hand, an improved quality of the harvestable products. In order to express foreign genes efficiently in plants, regulation signals which allow ordered transcription are required. These include promoters and terminators. The terminators located on the 3' end of the encoding DNA serve to end transcription and, if appropriate, as a signal for polyadenylation of the mRNA formed. Promoters contain recognition sequences for RNA-polymerases and for transcriptional effectors. The promoters are responsible for the expression behavior of the foreign genes.
Herbicide-tolerant plants as they are disclosed in DE-A-3701623 are an example of genetic engineering measures in crop protection. Other examples are insect-resistant plants (Vaek et al. (1987) Plant Cell 5, 159-169), virus-resistant plants (Powell et al. (1986) Science 232, 738-743) and ozone-resistant plants (Van Camp et al. (1994) BioTech. 12, 165-168). Examples of quality improvements achieved by genetic engineering are: better keeping qualities in fruit (Oeller et al. (1991) Science 254, 437-439), increased starch production in potato tubers (Stark et al. (1992) Science 242, 419), altered starch (Visser et al. (1991) Mol. Gen. Genet. 225, 289-296) and lipid composition (Voelker et al. (1992) Science 257, 72-74) and production of plant-foreign polymers (Poirer et al. (1992) Science 256, 520-523).
A large number of promoters which control expression of foreign genes in plants is known. The most frequently used promoter is the 35S CaMV promoter (Franck et al. (1980) Cell 21, 285-294).
This promoter contains various recognition sequences for transcriptional effectors which, in their totality, lead to constitutive expression of the gene which has been introduced (Benfey et al. (1989) EMBO J. 8, 2195-2202). Frequently, inducible or cell- or tissue-specific promoters are also employed.
Examples of inducible expression which have been described are, inter alia, the following: wound induction (DE-A-3843628, DE-B-3837752), chemical induction (Ward et al. (1993) Plant Molec. Biol. 22, 361-366) and light induction (Fluhr et al. (1986) Science 232, 1106-1112).
DE-A-4207358 discloses a promoter which causes gene expression specific to the stomatic cells, but no specific expression in mesophyll cells or in epidermal cells of leaves. An artificial change of the opening periods of the stomata allows the gas exchange of plants manipulated in this fashion to be regulated as desired. Herbicide tolerance or herbicide resistance can not be mediated by such a promoter.
Other examples of cell- and tissue-specific expression are: seed-, tuber- and fruit-specific expression (compiled in Edwards and Coruzzi (1990) Annu. Rev. Genet. 24, 275-303; DE-A-3843627), phloem-specific expression (Schmulling et al. (1989) Plant Cell 1, 665-670), root-nodule-specific expression (DE-A-3702497) and meristem-specific expression (Ito et al. (1994) Plant Molec. Biol. 24, 863-878). Examples of promoters in chloroplast-containing cells are also known from Edwards and Coruzzi (1990), Annu. Rev. Genet. 24, 277-279. The promoters described in this publication cause expression either only in inducible form (for example the rbcS-3A promoter) or only in certain types of cells (for example the GS2 and GS3A promoters), but the expression is not limited to certain parts of the plant.
The use of the above-described promoters is frequently problematic. For example, promoters which cause constitutive expression of the genes under their control can be employed for generating herbicide-tolerant and pathogen-resistant plants, but have the disadvantage that the products of the genes under their control exist in all parts of the plant, including the parts of the plant which are harvested, which may be undesirable in some cases. Equally, inducible promoters are not without problems since the conditions for induction in crop plants are typically difficult to control in the field.
In C3 plants, the promoter of phosphoenol pyruvate carboxylase, which originates from a C4 plant, leads to expression in the mesophyll of the leaf (Stockhaus et al., (1994), Mol. Gen. Genet. 245, 286-293). However, this promoter only mediates a low activity level in the mesophyll. Moreover, it also shows activity in roots. This poor organ specificity is undesired for many applications.
Promoters which cause leaf-specific, preferably permanent, expression of genes under their control have not been disclosed.
It would therefore be desirable to find routes to express genes in plants while avoiding the above disadvantages.